The novel neuropeptide spexin (Spexin/NQ/NPQ/SPX), which is encoded by the C12orf39 (chromosome 12 open reading frame 39) gene, was originally discovered using bioinformatics tools. The mature spexin peptide sequence consists of 14 amino acids formed as a result of cleavage of dibasic amino acids by a proprotein convertase and is very well conserved in typical vertebrate species as well as humans [Mirabeau et al., Genome Res, 2007, 17:320-327]. Spexin expression at the mRNA and/or protein level has been documented in brain regions and peripheral tissues of several species such as humans, mice, and goldfish, suggesting multiple physiological functions of spexin in vertebrates. Recently, spexin was implicated in regulation of feeding behaviors and related metabolic processes. Spexin mRNA levels are markedly decreased in the fat of obese humans, and administration of spexin leads to weight loss in diet-induced obese rodents. Spexin also suppresses appetite in goldfish. Recent reports have shown that spexin stimulates intestinal muscle contraction to induce bowel movements, is expressed in human endocrine and epithelial tissues, and is associated with glycometabolism and lipometabolism from its reduced level in patients with type 2 diabetes. In addition, spexin is likely involved in reproduction, cardiovascular/renal function, and nociception. [Waleski et al., Obesity 2014, 22:1643-1652; Wong et al., Am J Physiol Endocrinol Metab, 2013, 305:E348-366; Liu et al., Mol Cel Endocrinol, 2013, 374:65-72; Toll et al., FASEB J, 2012, 26:947-954; Lin et al., Sci Rep, 2015, 5:12095; Gu et al., Peptides, 2015, 71:232-239]. The precise roles of spexin in these processes, however, are not well understood due to a lack of information on the spexin receptor. Recently, the present inventors demonstrated that spexin is an endogenous ligand that acts at galanin receptor (hereinafter referred to as “GALR”) types 2 and 3 but not at GALR1, while galanin activates all three receptor subtypes. Indeed, it can be considered that galanin shares activity on GALR2 in common with spexin because of its very low potency for GALR3 [Kim et al., Endocrinology, 2014, 155:1864-1873].
The spexin and galanin genes likely emerged through a local duplication from a common ancestor gene, and as a result, their mature peptides share several conserved residues, including Trp2, Thr3, Tyr9, and Gly12 [Kim et al., Endocrinology, 2014, 155:1864-1873]. Like spexin, galanin is widely expressed in the central nervous system and peripheral tissues. The actions of spexin and galanin in appetite behavior and reproduction, however, appear to oppose each other. For instance, levels of galanin are significantly higher in obese women, and galanin administration or overexpression in genetically engineered mice results in an increase in food intake. Thus, galanin appears to be orexigenic, while spexin is anorexic. Administration of galanin-like peptide stimulates luteinizing hormone (LH) secretion, while spexin administration attenuates LH secretion in the goldfish [Barnowska et al., Metabolism, 1997, 46:1384-1389; Rada et al., Alcohol, 2004, 33:91-97; Castellano et al., Am J Physiol Endocrinol Metab, 2006, 291:E1281-1289; Liu et al., Mol Cel Endocrinol, 2013, 374:65-72]. These opposing effects are likely due to GALR receptor subtype-specific signaling pathways. Specifically, GALR1 and GALR3 induce inhibitory Gi-coupled signaling, while GALR2 triggers stimulatory Gq-coupled signaling [Webling et al., Front Endocrinol, 2012, 3:146].
Studies on GALR2-mediated phenotypes have been made through attempts to develop receptor gene knockout (KO) mice and agonists and antagonists selectively acting on the receptor. In GALR2 KO mice, no unusual abnormalities with respect to sensory function, feeding behavior, reproduction, mood, learning and memory were reported. Later then, anxiety- and depression-related behaviors were demonstrated in GALR2 KO mutants. This phenotype was similar to that observed in GALR1 KO mice; however, this GALR2-mediated effect is likely the opposite of the GALR3 effect, as GALR3-specific antagonists decrease anxiety and induce depression-like behavior [Gottsch et al., Mol Cell Biol, 2005, 25:4804-4811; Bailey et al., Pharmacol Biochem Behav, 2007, 86:8-20; Lu et al., Neuropeptides, 2008, 42:387-397; Holmes et al., Neuropsychopharmacology, 2003, 28:1031-1044; Swanson et al., Proc Natl Acad Sci USA, 2005, 102:17489-17494]. GALR2 deficiency resulted in developmental loss of dorsal root ganglion neurons and microinjection of a GALR2-specific agonist into the spinal cord induced allodynic effects, suggesting a possible role in pain behavior. Involvement of GALR2 in the mesolimbic reward system has been reported; galanin decreases the amplitude of excitatory postsynaptic potential in dorsal striatum and nucleus accumbens, and this effect is absent in GALR2 KO mice. In the central amygdala, galanin, through binding of the GALR2, decreases the amplitudes of GABAergic inhibitory postsynaptic potentials. Activation of GALR2 protects the hippocampus from neuronal damage through the phosphorylation of the serine/threonine kinase Akt [Shi et al., Eur J Neurosci, 2006, 23:627-636; Liu et al., Proc Natl Acad Sci USA, 2001, 98:9960-9964; Einstein et al., Eur J Neurosci 2013, 37:1541-1549; Bajo et al., Addict Biol, 2012, 17:694-705; Elliotte-Hunt et al., J Neurochem, 2007, 100:780-789]. Such GALR2-mediated phenomena function in opposition, in concert, or independently of GALR1 and GALR3-mediated phenomena. Nevertheless, these observations suggest the possible involvement of GALR2s in learning and memory, pain, anxiety, and mood disorders.
Several attempts have been made to develop GALR receptor subtype-specific agonists. A galanin fragment consisting of the amino acid residues at positions 2-11 of galanin, GAL (2-11), was first developed as a GALR2 selective agonists, but further studies unfortunately revealed that this fragment has similar affinity for the GALR3. Several GALR2-specific agonists, which were generated by modification at the N-terminus and/or C-terminus of galanin, have been reported over the years. Of these, M1145 and M1153 were found to exhibit GALR2 selectivity with 50-100-fold binding preference for GALR2 compared to GALR1 and GALR3; however, at high concentrations, these agonists retain substantial affinity for GALR1 and GALR3 [Liu et al., Proc Natl Acad Sci USA, 2001, 98:9960-9964; Lu et al., Neuropeptides, 2005, 39:165-167; Webling et al., Front Endocrinol, 2012, 3:146; Runesson et al., Neuropeptides, 2009, 43:187-192; Saar et al., Neurochrm Int, 2011, 6:714-720].
Despite numerous efforts to develop several species of galanin receptor subtype-specific agonists in the previous studies, treatment with high concentrations of the agonists was found to cause specificity problems. Under these circumstances, there arises a need to elucidate GALR subtype-specific mediated physiological functions in the development of receptor subtype-specific agonists.
Thus, the present inventors have succeeded in developing improved agonists by identifying the reactivity between spexin and GALR2 through sequencing and substitution of the constituent amino acids of galanin and spexin and increasing the stability of the agonists, and in elucidating the physiological functions of the agonists when administered to animals, achieving the present invention.